The present invention relates to the field of site-specific conjugation of synthetic particles to proteins. Synthetic particles include monodisperse synthetic particles, highly monodisperse nanoscale particles, and dendritic polymers (herein after dendrimers). Proteins include four-helical bundle proteins and cytokines. The present invention also relates to both novel methods of conjugation of synthetic particles to proteins and the resulting compositions.
Biotechnology has made the production and engineering of proteins a straight-forward commercially feasible technology. Recombinant proteins are linear amino acid polymers, synthesized from amino to carboxy ends, that fold into three dimensional configurations during or after synthesis. Biotechnology allows convenient production of individual protein molecules in bulk. Bioconjugate technology can add an additional dimension to protein structures by allowing higher-order organization of the individual proteins using a synthetic framework. Advantages of higher order structures containing proteins include, under some circumstances, therapeutically important properties including, but not limited to, circulating half-life, tissue or intracellular targeting, biodistribution, protein stability, ligand potency/activity, and protein immunogenicity. Regulating the relative positions and stoichiometries of individual proteins in higher order structures can also give rise to wholly new activities and functions.
PEG (polyethylene glycol) is a synthetic material commonly attached to proteins. A variety of means have been used to attach PEG to proteins. The most frequent method of attachment is through the amino groups as found on the lysine residues or at the N-terminus (See PCT Application WO 96/11953, incorporated by reference).
Kinstler et al., WO 96/11953 teaches that conjugation of PEG to a particular protein, namely G-CSF, by conventional means has deficiencies related to the poor site-specificity of the PEG conjugation. Kinstler identified that although the prior art provided multiple methods of conjugating PEG to proteins, none of the methods known in the art allowed for selective attachment. As noted above, selective attachment is desirable for many reasons including retention of protein bioactivity. Kinstler identified a method of bioconjugation to selectively attach PEG to the N-terminus of G-CSF. The method disclosed in Kinstler is specific for the conjugation of G-CSF to PEG. It is not contemplated by Kinstler, nor those skilled in the art, that the method used to conjugate G-CSF to PEG can be used to conjugate other proteins or to use different synthetic particles and materials while retaining particular properties of the protein.
Thus, there is a need for a more universal method of conjugation and more particularly the use of better methods of conjugating proteins to monodisperse, well-defined synthetic particles.
In its broadest sense, the present invention is directed to orthogonal chemistry for site specific conjugation/ligation of synthetic particles to the N-terminus of proteins. Synthetic particles include monodisperse synthetic particles, highly monodisperse nanoscale particles, and dentritic polymers (herein after dendrimers). Proteins include four-helical bundle proteins and cytokines.
Dendrimers, a subset of highly monodisperse nanoscale particles (also known as dense star polymers), offer benefits that other carriers known in the art lack. In particular, dendrimers exhibit molecular architecture characterized by regular dendritic branching with radial symmetry. See U.S. Pat. No. 5,527,524 incorporated by reference. This uniform architecture is desirable for homogenous ligated compositions. In addition, each dendrimer can be molecularly tailored to meet specialized end uses by controlling the size, shape and properties of the dendrimer.
Cytokines are small proteins that engage cell-surface receptors to elicit their biological activities. Synthetic particles are any particulate materials produced using synthetic chemical means. Monodisperse synthetic particles are any particulate materials produced using synthetic chemical means that are limited to a single chemical composition, size and architecture. Monodisperse nanoscale particles are any particulate materials produced using synthetic chemical means that are limited to a single chemical composition, size and architecture and which measure between 1 and 999 nanometers in each dimension (ie., length, width and depth).
One method of conjugation contemplated by the present invention entails coupling of sulfhydryl-terminated synthetic particles with a maleimide spacer on the protein. Another contemplated method is based on using a serine end-terminal (amine end) on the protein that can then be oxidized using periodate to form an aldehyde. The aldehydic protein can then be coupled to the synthetic functionalized with an aminooxyacetyl group to form a stabilized oxime. A further method to achieve essentially the same result is to couple an amine-terminated synthetic particle to the aldehydic protein. The Schiff""s base that is formed can then be stabilized using a mild reducing agent such as sodium cyanoborohydride.
One type of specialized synthetic particles are dendrimers. Dendrimers are polymers that are unimolecular assemblages possessing: 1) an initiator core; 2) interior layers (referred to as generations or G) made up of repeating units, radially attached to the initiator core; and 3) exterior surface of terminal functionality or terminal functional groups attached to the outermost generation. The size and shape of the dendrimer and the resulting functionality can be controlled by the choice of the initiator core, the number of generations and the choice of the repeating units employed at each generation. Since dendrimers can be isolated at any particular generation, dendrimers can be obtained having only the desired structural properties. (See U.S. Pat. No. 5,527,524, incorporated by reference). Particular methods of producing dendrimers can be prepared according to methods described in U.S. Pat. No. 4,587,329, incorporated by reference.
PAMAM (polyamidoamine) dendrimers are microdomains which very closely mimic classical spherical micelles in shape, size, number of surface groups and area/surface groups. A significant difference between micelles and PAMAM dendrimers is that PAMAM dendrimers are covalently fixed and robust compared to the dynamic equilibrating nature of micelles. This difference provides an advantage for the PAMAM dendrimers especially when using them as encapsulation devices. This advantage is most appreciated when the PAMAM dendrimers remain in the fifth generation or less. Generations more than five may cause congestion at the surface.
Dendrimers suitable for this invention include those described in U.S. Pat. Nos. 4,507,466, 4,558,120, 4,568,737, 4,587,329, and 5,527,524, incorporated by reference.
Although all synthetic particles are contemplated by this invention, monodisperse synthetic particles are more preferred. A more preferred embodiment of the invention uses highly monodisperse nanoscale particles. An even more preferred embodiment uses PAMAM dendrimers.
Proteins for this invention are either joined directly to the synthetic particle or in an alternative embodiment attached via a linker. In order to prepare the protein for the linker type application a GMBS-linker may be employed. A commercially obtained (Pierce, Rockford, Ill., USA ) sulfo-GMBS (N-gamma-maleimidobutyryloxyl sulfosuccinimide ester)linker sequence was appended to the N-terminal alanine of the protein at a high pH to achieve N-terminal site specificity of GMBS linker attachment. For the non-linker/direct attachment method, proteins were engineered by standard biotechnology methods to contain a serine at the N-terminus.
Although all proteins can potentially be utilized for this invention, a more preferred embodiment uses four-helical bundle proteins. An even more preferred embodiment uses cytokines. The proteins used for this invention can be natural or recombinantly produced. A more preferred embodiment uses recombinant proteins engineered for particular purposes or bioactivity.
It is also contemplated that the synthetic particle or more specifically a dendrimer can be engineered to be ligated to more than one protein.
A preferred method for site specifically attaching a synthetic particle to the N-terminus of a protein comprises the steps of:
1) attaching a spacer on the N-terminus of a protein;
2) forming a sulfhydryl on a synthetic particle at the amine; and
3) combining said sulfhydrylized synthetic particle to said spacer on the protein.
A more preferred method for site specifically attaching a synthetic particle to the N-terminus of a protein comprises the steps of:
1) converting a ser-terminated protein to an aldehyde;
2) converting the amine of a synthetic particle to a oxiamine; and
3)combining said aldehyde ser-terminated protein to said synthetic particle at said oxiamine.
The following three schemes describe processes of preparing novel conjugates. It would be obvious from the schemes to those skilled in the art that alternate reagents and modifications of the processes could be used depending on circumstances. These schemes are not intended to limit the scope of the invention. 
The following Examples are provided to illustrate the present invention and are not intended to limit the scope thereof. Those skilled in the art will readily understand that known variations of the conditions and processes of the following preparative procedures can be used to prepare these conjugates.
Without further elaboration, it is believed that one skilled in the art can, using the preceding descriptions, utilize the present invention to its fullest extent. Therefore, the following preferred specific embodiments are to be construed as merely illustrative and not limitative of the remainder of the disclosure in any way whatsoever. Compounds containing multiple variations of the structural modifications illustrated in the preceding schemes or the following Examples are also contemplated.
The starting materials which are required for the above processes herein described are known in the literature or can be made by known methods from known starting materials.